1. Field of the Invention
The present invention relates generally to microfluidic chips and methods for performing electrodeless purification, concentration, trapping and launching of polarizable particles or molecules in fractionating devices or chemical/amplification/detection devices by dielectrophoresis. The novel devices utilize dielectrophoresis technology achieved by using insulating constrictions without the use of metal electrodes and exploiting low frequency polarizability of particles or molecules. In particular, the polarizable particles and molecules include but are not limited to, cells, viruses, polymer particles, colloids and molecules such as proteins, peptides, carbohydrates, and polynucleotides, in particular, single-stranded or double-stranded DNA or RNA. The invention also relates to a device for thermocycling polarizable particles, in particular for amplification of nucleic acids. Specifically, the invention involves trapping minute amounts of nucleic acids in a microfabricated, dielectrically focused device, thermocycling them, and releasing them for fractionation or analysis.
2. Description of the Related Art
One of the great challenges in biotechnology is how to move and concentrate molecules in a micro-fabricated environment. One possible technique is dielectrophoresis (DEP) in which the translation of neutral matter is caused by polarization effects in a nonuniform electric field, see Polh, H. A., Dielectrophoresis: The Behavior of Neutral Matter in Nonuniform Electric Fields, Cambridge University Press, Cambridge, UK, (1978) and Pethig, R., Dielectrophoresis: Using Inhomogeneous AC Electrical Fields to Separate and Manipulate Cells, Crit. Rev. Biotechnol. Vol. 16, Iss. 4, pp. 331-348, (1996).
DEP has been used for sample manipulation at the molecular level. Applications of DEP include: separation of colloidal particles, as described in Rousselet, J. et al., Directional Motion of Brownian Particles Induced by a Periodic Asymmetric Potential, Nature (London), 370, pp. 446-448, (1994) and Green, N. G. et al., Dielectrophoresis of Submicrometer Latex Spheres Experimental Results, J. Phys. Chem. B, Vol. 103, Iss. 1, pp. 41-50, (1999); DEP ratchet, as described in Gorre-Talini, L. et al., Dielectrophoretic Pratchets, Chaos 8: (3) pp. 650-656 (September 1998); DEP coating, as described in Choi, W. B. et al., Field Emission from Silicon and Molybdenum Tips Coated with Diamond Powder by Dielectrophoresis, Appl. Phys. Lett., Vol. 68, Iss. 6, pp. 720-722, (1996); separation of yeast, as described in Markx, G. H. et al., Separation of Viable and Non-Viable Yeast Using Dielectrophoresis, J. Biotechnol. 23:29-37; separation of virus, as described in Morgan, H. et al., Separation of Submicron Bioparticles by Dielectrophoresis, Biophys. J. 77: pp. 516-525, (1999); separation of cancer cells, as described in Becker, F. F. et al., Separation of Human Breast-Cancer Cells from Blood by Differential Dielectric Affinity, Proc. Nat. Acad. Sci. (USA), Vol. 92, Iss. 3, pp. 860-864, (1995) and Yang, J. et al., Cell Separation on Microfabricated Electrodes Using Dielectrophoretic/Gravitational Field Flow Fractionation, Anal. Chem., Vol. 71, Iss. 5, pp. 911-918, (1999); and trapping and manipulation of DNA, as described in Washizu, M. et al., Electrostatic Manipulation of DNA in Microfabricated Structures, IEEE Trans. Ind. Appl., 26: pp. 1165-1172, (1990), Washizu, M. et al., Molecular Dielectrophoresis of Biopolymers, IEEE Trans. Ind. Appl. 30:835-843, (1994), Washizu, M. et al., Applications of Electrostatic Stretch-and-Positioning of DNA, Vol. 31, pp. 447-456 (1995), and Asbury, C. L. et al., Trapping of DNA in Nonuniform Oscillating Electric Fields, Biophysical Journal, 74:1024-1030 (1998).
The essence of dielectrophoretic trapping is that a dielectric object will be trapped in the regions of high field gradient provided there is sufficient dielectric response to overcome thermal energy and the electrophoretic force. A conventional method to make a DEP trap is to create an electric field gradient by an arrangement of fine planar electrodes either: directly connected to a voltage source; as described in Rousselet, J., et al., Directional Motion of Brownian Particles Induced by a Periodic Asymmetric Potential, Nature (London), 370, 446-448, (1994) and Green, N. G. et al., Dielectrophoresis of Submicrometer Latex Spheres Experimental Results, J. Phys. Chem. B, Vol. 103, Iss. 1, pp. 41-50, (1999); or free floating, as described in Washizu, M. et al., Molecular Dielectrophoresis of Biopolymers, IEEE Trans. Ind. Appl. 30:835-843, (1994); Washizu, M. et al., Applications of Electrostatic Stretch-and-Positioning of DNA, Vol. 31, pp. 447-456 (1995); and Asbury, C. L. et al., Trapping of DNA in Nonuniform Oscillating Electric Fields, Biophysical Journal, 74:1024-1030 (1998).
U.S. Pat. No. 6,117,660 describes a method of treating material with electrical fields and with an added treated substance. A plurality of electrodes are arrayed around the material to be treated and are connected to outputs of an electrode selection apparatus. Inputs of the electrode selection apparatus are connected to outputs of an agile pulse sequence generator. A treating substance is added to the membrane-containing material. Electrical pulses are applied to the electrode selection apparatus and are routed through the electrode selection apparatus in a predetermined, computer-controlled sequence to selected electrodes in the array of electrodes, whereby the membrane containing material is treated with the added treating substance and with electrical fields of sequentially varying directions.
U.S. Pat. No. 6,071,394 describes a method for performing channel-less separation of cells by dielectrophoresis, lysis and diagnostic analyses. A cartridge including a microfabricated silicon chip on a printed circuit board and a flow cell mounted to the chip forms a flow chamber. The cartridge also includes output pins for electronically connection the cartridge to an electronic controller. The chip includes a plurality of circular microelectrodes which are preferably coated with a protective permeation layer which prevents direct contact between an electrode and a sample introduced into the flow chamber and enables immobilization of specific antibodies for specific cell capture. The amplification of nucleic acids is central to the current field of molecular biology. Library screening, cloning, forensic analysis, genetic disease screening and other increasingly powerful techniques rely on the amplification of extremely small amounts of nucleic acids. As these techniques are reduced to a smaller scale for individual samples, the number of different samples that can be processed automatically in one assay expands dramatically. For further improvements, new integrated approaches for the handling and assaying of a large number of small samples are needed.
With the polymerase chain reaction (PCR) for nucleic acid amplification, a purified DNA polymerase enzyme is used to replicate the sample DNA in vitro. This system uses a set of at least two primers complementary to each strand of the sample nucleic acid template. Initially, the sample nucleic acid is heated to cause denaturation to single strands, followed by annealing of the primers to the single strands, at a lower temperature. The temperature is then adjusted to allow for extension of the primers by the polymerase along the template, thus replicating the strands. Subsequent thermal cycles repeat the denaturing, annealing and extending steps, which results in an exponential accumulation of replicated nucleic acid products.
PCR represents a considerable time savings over the replication of plasmid DNA in bacteria, but it still requires several hours. PCR also has limitations in the subsequent handling of the product. Most reactions occur isolated in a test tube or plate containing the require reagents. Further analysis of these products entails removing them from the tube and aliquoting to a new environment. Significant delay and loss and damage to the product may result from such a transfer. Emerging technology in the display of nucleic acids in arrays on chips, for further identification and selection, requires a more precise method of transfer of samples from amplification step to chip than is possible by dispensing the contents of each reaction tube individually.
Another disadvantage of PCR is the requirement of a reaction tube which has a volume possible too large for the amplification of particularly minute amounts of starting template and other reagents. PCR is able to amplify just one molecule of template, but the volume of the reaction mixture makes this goal difficult to achieve. The nature of the reaction tube also requires a signification volume of the other reagents. Conventional PCR involves heating and cooling the reaction tube several times for each cycle, requiring elaborate instrumentation to control the temperature of the apparatus which holds the tubes, and the tubes and solutions they contain, over time.
Lab-on-a-chip or biochip technology for manipulating DNA on a small scale is a recent development in the art. For example, Austin et al., U.S. Pat. No. 5,427,663, describes a microlithographic array for fractionating macromolecules. Heller, U.S. Pat. No. 5,605,662 describes a microfabricated device having DC microelectrodes for DNA hybridization. Individual wires in a direct electrophoretic field have been used to focus and launch DNA into separation media.
Microfabricated devices can be used for PCR. Wilding, et al. (Clin.Chem. 40:1815-8, 1994) designed a photolithographed, sealed silicon chip which can receive reagents by capillary action and can be mounted on a Peltier heater-cooler. This design reduces reagent volumes, but requires an external source for heating and does not couple positioning and manipulation of the nucleic acids, and so amplification and subsequent analysis on a fractionation matrix cannot be achieved without transfer of samples. Thus, prior art systems for the amplification of nucleic acids do not integrate micromanipulation and amplification steps, such that the need for transfer steps reduces the quantity and quality of the products, and time and labor are increased.
In another field, dielectrophoresis has been used to position cells and molecules on a micron scale. Washizu and Kurosawa, IEEE Transactions on Industry Applications, 26(6): 1165-1172, 1990, and Washizu et al., IEEE Transactions and Industry Application, 31(3): 447-455, 1995, used high voltage (104 V/cm) and high frequency (1 MHz) alternating currents to generate a dielectric field in a micron-sized floating electrode. Alignment, permanent fixation, stretching, and cutting of DNA molecules was described. Asbury and van den Engh (Biophys. J. 74:1024-30, 1998) report a sealed device having an array of 100 strips of gold which, when placed in an oscillating field, reversibly traps DNA along the edges of the strips.
U.S. Pat. No. 6,051,380 describes a self-addressable, self-assembling microelectronic device designed and fabricated to actively carry out and control multi-step and multiplex molecular biological reactions in microscopic formats. The device can subsequently control the transport and reaction of analytes or reactants at the addressed specific microlocations. The device is able to concentrate analytes and reactants, remove non-specifically bound molecules, provide stringency control for DNA hybridization reactions and improve the detection of analytes. The device has an array of electronically programmable and self-addressable microscopic locations. Each microscopic location contains an underlying working direct current (DC) or DC/AC microelectrode supported by a substrate. The surface of each microlocation has a permeation layer for the free transport of small counter-ions, and an attachment layer for the covalent coupling of specific binding entities. The device has a matrix of addressable microscopic locations on its surface. Each individual micro location is able to electronically control and direct the transport and attachment of specific binding entities (e.g., nucleic acids, antibodies) to itself. All microlocations can be addressed with their specific binding entities.
The above-described systems have the limitation that the use of metallic electrodes on the microchip requires complex manufacturing steps such as metal evaporation during fabrication. There is a need to develop an effective technique for moving and concentrating polarizable particles without the use of metal electrodes.
The present invention further provides a device for the integrated micromanipulation, amplification, and analysis of polarized particles such as DNA comprises a microchip which contains constrictions of insulating material for dielectrophoresis powered by an alternating current or direct current signal generator, and attached to a hot source that can be heated to specific temperatures. Nucleic acids can be heated and cooled to allow for denaturation, and the annealing of complementary primers and enzymatic reactions, as in a thermocycling reaction. After such a reaction has been completed at the constriction, the dielectrophoretic field can be switched to a direct field to release the product and direct it through a matrix for fractionation. The device includes data analysis equipment for the control of these operations, and imaging equipment for the analysis of the products. The invention permits the efficient handling of minute samples in large numbers, since reactions occur while sample material is trapped between constrictions. Because the positioning, reactions, and release into a fractioning matrix all occur at the constriction which serves as a focusing locus, the need to transfer samples into different tubes is eliminated, thus increasing the efficiency and decreasing the possibility of damage to the samples.
This invention relates to a microfluidic device for trapping nucleic acids by dielectrophoresis, thermocycling them on the electrode, and then releasing them and fractionating through a gel, or other medium, for analysis. The invention avoids the need for an external thermocycling device, reduces the volume and amount of starting materials and reagents, and reduces the time and manipulations needed to complete an amplification protocol and sequencing.
This arrangement improves prior nucleic acid amplification steps by decreasing the required time and reagent volume. The entire apparatus is contained on a monolithographic wafer. Because the reactions take place in such a small volume, and the nucleic acid templates are positioned directly on the actual heat source, as opposed to in a tube isolated from the heat source, the time for temperature changes to perform PCR is significantly reduced.
The use of an integrated device for dielectric focusing, to position the micromolecule templates for amplification and for subsequent analytical steps such as fractionation by size or sequencing, eliminates the need for transferring samples between these steps. When the samples are released from the constrictions, they can be electrophoresed through an adjacent matrix to achieve fractionation. These coupled reactions are suitable for multisample arrays, such as standard plates which are multiples of 96-sample arrangements.
This invention satisfies a long felt need for an integrated microfabricated device suitable for thermocycling of polarizable particles using minimal starting materials, in a minute volume, and permits amplification and fractionation of DNA without transfer.
The dimensions of the field electrodes are not critical so long as the field electrodes produce the desired dielectrophoretic field. Conventional gold electrodes may be used, as can other metals having the desired characteristics of inertness with respect to electrolysis of the electrode. The width or thickness of the electrodes can be in the range of about 100 xcexcm to about 1 mm, preferably from about 200 xcexcm to about 500 xcexcm, for example 250 xcexcm.
The substrate chip is typically quartz or silicon dioxide, for ease of manufacture and transparency suitable for microscopic examination, but other materials and composites now known or later discovered could be used, for example glass, silicon nitride, or polymers. The constrictions may be silicon dioxide, polymide, PMMA or other suitable inert materials that can be deposited or machined with the requisite accuracy.
The cover for the chip may be glass, quartz, polymers or other suitable material, preferably transparent at least in the regions where observation of the microchannels is required. The cover may be integral or removable, depending primarily on the manufacturing process.
In the most preferred embodiment, the polarizable particle is DNA, while in other embodiments it might be a protein, or other biological or synthetic polymer. With DNA, the buffer solution preferably contains suitable salts and buffers, such as xc2xdxc3x97TBE, TAE, MOPS, SDS/Tris/glycine, or TAPS.
For microreactions with polarizable particles other than nucleic acids, the material can be trapped at the constrictions by dielectrophoresis, subjected to desirable microreactions, including thermal cycling as needed, and then released and fractionated. For example, a polymerase protein may be focused and trapped together with nucleic acid to facilitate polymerization.
In summary, the invention relates to a device for selectively trapping, thermocycling, and releasing polyelectrolytes, comprising: (a) a microlithographic substrate having a microfluidic channel dimensioned to accommodate a fluid, (b) field electrodes positioned to provide a dielectrophoretic field along the channel in response to an alternating current, (c) constrictions positioned between the field electrodes, the field electrodes and constrictions being capable of fluid communication with each other via the channel, and (d) circuitry controlling current to the field electrodes, whereby a polarizable particle in solution may be (i) trapped at the constrictions when an alternating field is applied to the field electrodes, (ii) heated when a field is applied to the constriction, and (iii) released when the trapping alternating field is not applied.
A method for thermal cycling of a polarizable particle comprises: (a) placing the polarizable particle in solution in a channel adjacent to a constriction, (b) trapping the polarizable particle between contstrictions by applying a dielectrophoretic field to the solution, and (c) releasing the polarizable particle by removing the dielectrophoretic field, and further heating the polarizable particle by applying a current to a resistor or peltier, and removing the current and allowing the nucleic acid to renature. The polarizable particle may be nucleic acid, denatured on heating.
The method may further comprise determining the sequence of the nucleic acid by reacting the trapped nucleic acid with amplification reagents, allowing nucleic acid amplification to occur, releasing the polarizable particle by removing the dielectrophoretic field, fractionating the nucleic acid, and scanning the fractions. A plurality of samples can be processed simultaneously in separate devices. The method can include fractionating and/or analyzing the released polarizable particle by electrophoresis such as in a field produced by the field electrodes.